The team set out to map where two antimalarial compounds, PRC1590 and quinine, accumulate inside Plasmodium falciparum, the deadliest human malaria parasite. Both compounds are intrinsically fluorescent, meaning their aromatic chemical structures emit light under ultraviolet illumination without any chemical tagging. This is a genuine advantage. Attaching a synthetic fluorophore to a small drug molecule demands extensive medicinal chemistry and can alter how the compound behaves, potentially changing the very mechanism researchers are trying to study. Using intrinsic fluorescence seems to sidestep that problem entirely, and the approach has been used successfully before, for example with diamidine compounds that were shown to co-localize with DNA in several parasites.

PRC1590, a tetrahydro-beta-carboline antimalarial, was a particularly intriguing candidate. The compound is most potent against the trophozoite stage of the parasite, and resistance to it is mediated by PfMDR1, a transporter embedded in the membrane of the digestive vacuole, the parasite’s acidic food compartment and the classic target of drugs like chloroquine. The researchers hypothesized that PRC1590 acts at the digestive vacuole and designed co-localization experiments to test the idea. Quinine served as a positive control, since an earlier study had used its intrinsic fluorescence to show overlap with LysoTracker Red DND-99, a dye that stains acidic organelles including the digestive vacuole.

At first glance, the experiment appeared to work. Quinine’s fluorescence did overlap with LysoTracker staining, replicating the published localization. PRC1590 also produced a fluorescent signal in the region of the digestive vacuole. But then the team did something that many published localization studies omit: they imaged untreated parasites and parasites treated only with the vehicle, DMSO, under identical conditions. The controls glowed almost exactly as brightly as the drug-treated parasites. A one-way ANOVA across the four conditions found no statistically significant difference in mean fluorescence intensity at 405 nanometers, with a p-value of 0.627 and a Cohen’s f of just 0.078, far below any threshold of practical significance. The apparent localization of both drugs was a false positive.

The same pattern emerged when the researchers repeated the experiment with a genetically engineered parasite line carrying a green fluorescent protein tag on Plasmepsin II, an enzyme naturally trafficked to the digestive vacuole. Protein fluorophores generally outperform chemical stains because they are brighter, so this should have been a cleaner test. Instead, untreated controls again showed 405-nanometer fluorescence indistinguishable from PRC1590-treated parasites, with no significant differences in either mean intensity or Pearson’s correlation coefficients. Under the tested conditions, the intrinsic fluorescence of PRC1590 simply could not be distinguished from the background glow of the parasite itself.

Two distinct technical culprits emerged from the data. The first is spectral overlap, also called emission crosstalk. When the excitation or emission spectrum of one fluorophore overlaps with another, signal bleeds through between detection channels, creating spurious correlations. Notably, the proPM II-GFP parasites, imaged at 488 nanometers, showed a higher correlation with the 405-nanometer channel than LysoTracker-stained parasites imaged at 561 nanometers, exactly as crosstalk theory predicts, since 488 is closer to 405. The second culprit is autofluorescence. Biological samples are full of endogenous fluorophores, including aromatic proteins, the coenzyme NADPH, lipids, and lipopigments, all of which emit light when excited at suitable wavelengths. The team detected parasite autofluorescence under multiple imaging regimes: live parasites stained with BODIPY membrane dye on a confocal microscope, and fixed parasites on a widefield Delta Vision II system, where illuminating the whole sample increases background fluorescence and makes autofluorescence more visible.

The implications reach well beyond one compound. In a literature survey, the authors found that many published studies of intrinsically fluorescent antiparasitic drugs do not report negative controls at all. Some demonstrate co-localization with multiple stains, or show images at varying drug incubation times and concentrations, but none of these substitutions replace a properly matched negative control. Only a single study in their search had imaged drug-treated uninfected red blood cells as a control. Even that approach, the authors argue, is inadequate, because the malaria parasite itself is autofluorescent, so absence of signal in uninfected host cells says nothing about background in infected ones. Parasite autofluorescence at around 320 nanometers has even been proposed as a diagnostic marker of infection, underscoring how bright the parasite’s intrinsic glow can be.

From their troubleshooting, the researchers distilled practical guidance for anyone attempting these experiments. First, they recommend measuring a compound’s excitation and emission spectra at imaging-relevant concentrations and across a range of pH values before touching a microscope, because acidity can shift fluorescence properties, and acidic organelles like the digestive vacuole could attract signal for purely chemical reasons. Second, untreated and vehicle-treated parasites must be imaged as negative controls under identical microscope settings, a requirement they found absent from most of the literature. Third, and perhaps most fundamentally, they caution that subcellular accumulation alone is not evidence of target engagement. Weak bases like chloroquine accumulate in the digestive vacuole through ion trapping, and the parasite remodels its host red blood cell with new permeability pathways that change drug uptake, so a drug may pile up somewhere without that being where it acts.

The authors also point to complementary strategies that can strengthen mechanism-of-action studies. Comparing digestive vacuole morphology between treated and untreated parasites, imaging parasites with different subcellular markers after drug exposure, resistance selection combined with in vitro evolution and whole-genome sequencing, protein stability assays, and chemoproteomics can all provide more decisive evidence than localization images alone. The team acknowledges the limits of their own work: a small number of compounds, a limited set of imaging settings, and a single Plasmodium species. Still, the message is likely to resonate across parasitology and drug discovery. Intrinsically fluorescent drugs remain an attractive shortcut for watching a molecule inside a cell, but as this study demonstrates, without rigorous, well-matched controls the microscope can happily show researchers exactly what they hoped to see.

Subject of Research: Imaging artifacts and false positive co-localization in fluorescence microscopy of intrinsically fluorescent antimalarials in Plasmodium falciparum

Article Title: Navigating imaging artifacts: challenges with visualizing intrinsically fluorescent antimalarials

Article References: Bremers, E. K., Anaguano, D., Carlier, P. R., Muralidharan, V., & Cassera, M. B. (2026). Navigating imaging artifacts: challenges with visualizing intrinsically fluorescent antimalarials. Frontiers in Parasitology, 5, Article 1919904. https://doi.org/10.3389/fpara.2026.1919904

Image Credits: AI Generated

DOI: 10.3389/fpara.2026.1919904

Keywords: malaria, Plasmodium falciparum, fluorescence microscopy, autofluorescence, spectral overlap, antimalarial drugs, PRC1590, quinine, digestive vacuole, co-localization, drug discovery, imaging artifacts